Polynucleotides for detecting leishmanias and method of detection of leishmanial protozoan

ABSTRACT

The present invention enables detection, and isolation of DNP, specific to leishmanias, and identification of the sequence thereof. 
     Accordingly, a leishmania protozoan can be precisely detected. The present invention can be applied to a diagnosis reagent, diagnosis kit, and the like for diagnosing infection with a leishmania protozoan. Furthermore, brazil type leishmanias and mexico type leishmania can be differentiated, whereby the present invention can be applied to development of a remedy after the diagnosis of the leishmania infection. The present invention can be applied also to development of a reagent, a diagnosis kit, and the like for large-group examination for examining quickly and simply a large number of samples at a time in leishmaniasis propagating area.

This application is the national phase of international applicationPCT/JP96/00893, filed Apr. 2, 1996 which designated the U.S.

TECHNICAL FIELD

The present invention relates to DNA specific to leishmania protozoans,and a method for detecting the leishmania protozoan by use of the DNA.

BACKGROUND ART

Leishmaniasis is a disease caused by infection with a protozoan ofLeishmania genus (see, for example, IKAGAKU DAIJITEN 49, ENCYCLOPEDIA OFMEDICAL SCIENCES, published in April 1993, Kodansha). More than12,000,000 people are estimated to be suffering from this disease.Leishmaniasis is one of the important diseases designated by WHO. Theleishmania protozoans are classified morphologically into two types:amastigote and promastigote. The both types of protozoans are known topromulgate as the promasitgote type when the protozoan is transplantedto an appropriate culture medium.

Three species are known to infect humans: Leishmania donovani,Leishmania tropica, and Leishmania braziliensis.

These three species have not been differentiated from each othermorphologically. However, in recent years, these species have come to beknown to include subspecies and varieties by immunological andbiochemical techniques.

The methods for detection of the pathogen include direct detection ofthe protozoan, and indirect estimation of infection with the protozoanby immune reaction or a like method.

Of these, the direct detection is sure, but requires long time andcomplicated operations, being not practicable in a short time and inlarge numbers.

The known indirect detection methods include Chopra's antimony reaction,Napier's aldehyde reaction, Branchari's euglobulin reaction, and soforth.

Further, complement-fixation reaction, indirect hemagglutinationreaction, and indirect immunofluorometry are known as the immunologicalmethods therefor.

The definite identification of the species is conducted by usingcultivated protozoan by Adler's test, measurement of buoyant-density ofkineplast DNA, isozyme analysis, and so forth as well as the immunereaction.

DISCLOSURE OF INVENTION

As explained above, the known methods of detection of the leishmaniaprotozoan are not practically useful in view of precision, sensitivity,and operation efficiency, and are not applicable to development of adiagnosis medicine and a diagnosis kit for group examination and thelike which are potent means for inhibiting the leishmaniasis infection,disadvantageously.

Moreover, although the decisive identification of the species isimportant for selection or establishment of treatment for the infection,no practically useful identification method is found.

With the above problems, the object of the present invention is toprovide a sure and quick method for judging leishmania infection andidentifying the species thereof in a large number, and to make possiblethe development of diagnosis medicines and diagnosis kits for groupexamination which is effective in inhibiting leishmania infection.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A shows electrophoresis patterns of DNA amplified by a PCR methodemploying an LS-3 primer.

FIG. 1B shows electrophoresis patterns of DNA amplified by a PCR methodemploying an LS-3 primer.

FIG. 2A shows that amplification of DNAs of the F4 band is observed inall kinds of leishmanias when F4 detection primers (SEQ ID NO:5 and 6)are employed.

FIG. 2B shows that amplification of DNAS of the F4 band is observed inall kinds of leishmanias when F4 detection primers (SEQ ID NO:5 and 6)are employed.

FIG. 3A shows that amplification of DNAs of the F10 band is observedonly in Leishmania braziliensis type when F10 detection primers (SEQ IDNO:2 and 3) are employed.

FIG. 3B shows that amplification of DNAs of the F10 band is observedonly in Leishmania braziliensis type when F10 detection primers (SEQ IDNO:2 and 3) are employed.

FIG. 4 shows that a trypanosome, a relative parasite, gives no amplifiedDNA when the F4 detection primers and the F10 detection primers areemployed.

BEST MODE FOR CARRING OUT THE INVENTION

The present invention is described below in detail by reference to theannexed drawings.

The present invention provides methods for detecting and isolating a DNAspecific to various leishmanias, for identifying the base sequencethereof, and for judging the leishmania infection and the kind of theleishmania in a test sample.

More specifically, the present invention relates to a polynucleotidecontaining characteristically, in the molecule, at least the basesequence SEQ ID NO:1:

CTGTGTTAAT CTCAGTCGTC CTTCTCTTCT CTTCTGACTG GCTCGGCGCT

CGGTACCGCT TCTCGTTTCG CTTTGAACGG GAGAGCGGAG GAGAACGAGG

AGGTGGGCGT ATCIGCTGAT GAGAGCGGTC GGATCTGCAT GCATCACCGG

TCCCTCGGAT GCACACACAT ACACACACAC TCGGCCCGCA GTCCCTCGCT

TTGTGCCGCC TTTTTTCTTG TCTTGCCTTA CGCCATGTAC TGCGACCACC

CACACACACA CAC

The present invention relates also to a polynucleotide containingcharacteristically, in the molecule, at least a base sequence capable ofhybridizing to the polynucleotide having the above base sequence.

The present invention relates further to a polynucleotide containingcharacteristically, in the molecule, at least a base sequence capable ofhybridizing to the polynucleotide having a base sequence complementaryto the above base sequence.

The present invention relates further to a polynucleotide or acomplementary polynucleotide comprising a sequence of at least 15 basesselected from the aforementioned three base sequences.

The present: invention relates further to a polynucleotide having a basesequence SEQ ID NO:2 of CTGACTGGCT CGGCGCTCGG TAC.

The present invention relates further to a polynucleotide having a basesequence SEQ ID NO:3 of GGTCGCAGAA CATGGCGTAA GG.

The present invention relates further to a polynucleotide having atleast a base sequence SEQ ID NO:4:

GTTCATGCAC GCCACTACTT GCAAGGGTCA CTCGGCATTT TGCGAGGATA

AAGGGAAAGA GTTGACATTG CGGCGGAGGT TAGACATGCA AGTCAGGGCA

CGGATGTGCG CCATCTCGTA CCCTG

The present invention relates further to a polynucleotide containingcharacteristically, in the molecule, at least a base sequence capable ofhybridizing to the polynucleotide having the above base sequence.

The present invention relates further to a polynucleotide containingcharacteristically, in the molecule, a base sequence capable ofhybridizing with the polynucleotide having a base sequence complementaryto the above base sequence.

The present: invention relates further to a polynucleotide or acomplementary polynucleotide comprising a sequence of at least 15 basesselected from the aforementioned three base sequences.

The present invention relates further to a polynucleotide having a basesequence SEQ ID NO:5 of GTTCATGCAC GCCACTACTT GCAAGG.

The present invention relates further to a polynucleotide having a basesequence of CAGGGTACGA GATGGCGCAC ATCC.

The present invention relates further to a method of detecting a DNAcoming from a leishmania protozoan or a polynucleotide derived from theDNA in a sample by amplifying a DNA coming from a leishmania protozoa bygene amplification reaction with a template of DNA derived fromleishmania protozoa contained in a sample and detecting the amplifiedDNA or a polynucleotide derived from the DNA.

Leishmania Protozoans, Kinds Thereof, and Cultivation Thereof

The seven kinds of leishmania protozoans relates to the presentinvention:

L. braziliensis panamensis (MHOM/PA/71/LS94)

L. braziliensis braziliensis (MHOM/BR/75/M2904)

L. braziliensis guyanensis (MHOM/BR/75/M4147)

L. mexicana mexicana (MNYC/BZ/62/M379)

L. mexicana aristedesi (MORY/PA/68/GML3)

L. mexicana amazonensis (MHOM/BR/73/M2269)

L. major (MHOM/SU/73/5ASKH)

L. chagasi (MHOM/BR/74/PP75)

Extraction of Gene (DNA)

The method of separation of a DNA from the above protozoan and themethod of purification thereof are not specially limited in the presentinvention, and can be conducted by any known method. In particular, thephenol/chloroform method can be suitably used in the present invention.

Primer for Arbitrarily Primed Polymerase Chain Reaction (AP-PCR Primer)

Gene amplification is conducted for detecting a DNA common to theleishmanias in the present invention. The arbitrary primer therefor isnot limited. For example, the present invention uses following sixprimers selected as arbitrary primers:

INV-1 (5'-ACCGGAATTCTTGGCTGTTGGCACGATGAG-3') SEQ ID NO:7

INV-2 (5'-TTGCAAGCTTTTATCTGGTTATATTGACAG-3') SEQ ID NO:8

LS-3 (5'-AAGTGTTGATACCCACTTTGT-3') SEQ ID NO:9

LS-2 (5'-CAATGGGTTACTGTTACAAC-3') SEQ ID NO:10

MS2-F (5'-CCTTAGGTTCTGGTAATGAC-3') SEQ ID NO:11

MS2-R (5'-GGGTGC,A.TCTCACTGGGAC-3') SEQ ID NO:12

These six primers are useful for detecting only the DNA common to theseven kinds of leishmania protozoans and the DNA specific to the braziltype leishmanias.

Investigation of Gene Amplification Reaction Conditions

The conditions for the gene amplification reaction are not speciallylimited, and may be established by reference to the method of Saiki, etal. (Saiki, et al., Science, 230, 1350, 1985; Saiki, et al., Science,239, 487, 1988), and published books (e.g., Molecular cloning, ColdSpring Harbor Laboratory Press, 2nd ed., 1989).

In the present invention, for example, the conditions below are suitablyemployed.

For the DNA polymerase chain reaction, the conditions below are suitablyemployed.

(1) one cycle of 94° C. for 5 min,

(2) 10 cycles of 94° C. for 1 min; 37° C. for 1 min; and 72° C. for 1min,

(3) 30 cycles of 94° C. for 1 min; 60° C. for 1 min; and 72° C. for 7min,

(4) one cycle of 72° C. for 7 min, and

(5) storage at 15° C.

Detection of Leishmania Protozoan DNA Fragment

The sample after the gene amplification reaction can be separated andanalyzed, for example, by a polyacrylamide gel electrophoresis (PAGE)(e.g., Molecular Cloning, Cold Spring Harbor Laboratory Press, 194-196,1982, 6.36), and the respective bands can be identified, for example, bydying by an ethidium bromide dying method (e.g., Molecular Cloning, ColdSpring Harbor Laboratory Press, 194-196, 1982, 6, Chapter 36).

The DNA common to leishmanias or the DNA specific to a special typeleishmania can be selected by comparing the electrophoresis patterns ofthe amplified DNAs.

For example, in the present invention, as shown in FIG. 1A and FIG. 1B,use of the LS-3 primer selected from the above 6 primers enablesamplification of a specific band of DNA (referred to as F4) in common toall the leishmanias, and amplification of another specific band of DNA(referred to as F10) in common to the brazil type leishmanias.

Sequencing of Bases of DNAs Specific to Leishmanias

The method of sequencing of the bases of the DNAs of the F4 band and F10band is not specially limited in the present invention. Known methodsincluding Maxam-Gilbert method, Sanger method, and DNA sequencer methodare applicable for the base sequencing. For example, a DNA sequencer,Model 373A manufactured by ABI, is useful. That is, the base sequencesof the above two kind of DNAs, F10 and F4, were determined by use of theDNA sequencer of Model 373A produced by ABI, obtaining the base sequenceshown in Sequence Tables 1 and 2.

Selection of Polynucleotide for Detection of DNA Specific to Leishmania

The polynucleotide primer (forward primer, and reverse primer) fordetecting the DNA specific to leishmania in a sample by geneamplification may be selected from the base sequences of theleishmania-specific DNA as determined above.

For example in the present invention, the polynucleotides having a basesequence shown in SEQ ID NO:5 and 6 may be selected for detection of theDNA band F4.

The polynucleotides having base a sequence shown in SEQ ID NO:2 and 3may be selected for detection of the DNA band F10.

The preparation method for the selected polynucleotide is not speciallylimited, and any known oligonucleotide preparation means is applicable.For example, the syn hesis can be conducted generally by use of anautomatic DNA synthesis machine, or a like means. In the presentinvention, the synthesis can be conducted by use of a 392DNA/RNAsynthesis machine manufactured by ABI Co.

Furthermore, the leishmania-specific DNA can be amplified by a varietyof gene amplification reaction by utilizing the above obtainedpolynucleotide primer for detection of leishmania-specific DNA.

Gene Amplification Reaction

In the present invention, by adding the detecting polynucleotide of thepresent invention, namely a primer, to the sample, the leishmania genecan be amplified by a primer elongation reaction if an objectiveleishmania protozoan is contained in the sample. In the presentinvention, the method of the gene amplification is not speciallylimited, and the elongation reaction may be conducted by a generalmethod in which four kinds of nucleotide triphosphate dNTP(deoxyadenosine triphosphate, deoxycytidine triphosphate,cdeoxythymidine triphosphate, and deoxyguanosine triphosphate) as thesubstrate are bonded to the polynucleotide primer.

The useful enzymes include DNA polymerase I of Escherichia coli (E.coli), Klenow fragment of E.coli DNA polymerase I, T4DNA polymerase, Taqpolymerase derived from heat-resistant bacteria, TthDNA polymerase, pfUpolymerase, and the like.

In particular, preferred are DNA polymerases derived from heat-resistantbacterias which retain their enzyme activity at a high temperature,since the specificity of the primer to recognize the base sequence ofthe objective DNA can be raised. (see Japanese Patent Laid-Open Nos.1-314965 and 1-252300 for the detail).

In the present invention, the DNA of the F10 band common to leishmaniascan be amplified efficiently by repeating the elongation reaction by useof combination of the primers shown in SED NO:5 and 6, and the DNA ofthe F4 band which is specific to brazil type leishmanias can beamplified efficiently by repeating the elongation reaction by use ofcombination of the primers shown in SEQ ID NO:2 and 3.

The reaction conditions of the gene amplification are selected accordingto published books, for example: Molecular Cloning 2nd ed., 1989, ColdSpring Harbor Laboratory Press chapter 4; and PCR Jikken Manyuaru, HBJShuppann-sha, Jikken Igaku (Experimental Medicine), Special Issue,Vol.8, No.9, 1990, Yodo-sha Co.

Test Samples and Detection Method

The sample to be tested is not limited in the present invention. The DNAband F4 specific commonly to 7 kinds of lehshmanias and the DNA band F10specific commonly to brazil type leishmanias can be detected by geneamplification with the aforementioned DNA-detecting polynucleotidespecific leishmanias.

The test sample can be taken, for example, from the tissue of peripheryof a focus judged from its appearance to be infected with leishmania.The sample can be taken, for example, according to the method describedin a published book: Byori Hyohon no Tsukurikata (Preparation ofPathology Specimen) (Bunnkodo).

For example, the tissue taken out is enclosed in a compound, quicklyfrozen by liquid nitrogen or the like, and sliced by a cryostat intofrozen section slides, and the obtained section slides can be subjectedto gene amplification reaction. In this case, the presence or absence ofthe gene coming from leishmania can be detected by use of the obtainedseveral slides.

The amplified gene is detectable by a general method. For example, it isseparated by agarose electrophoresis, and dyed with ethidium bromide orthe like, and the bands of the genes can be detected by ultravioletlight irradiation.

A sample may be a DNA extracted from a taken-out tissue (see, e.g., themethod described in Saibo Kogaku Jikken Purotokoru (Experiment Protocolin Cell Engineering) pp.5-7, Shujun-Sha).

That is, the Southern blot technique (Southern, E. M., J.Mol.Biol., 98,503, 1975) is applied (Molecular Cloning, Cold Spring Harbor LaboratoryPress, 2nd ed. 1989).

The DNA derived from the taken-out tissue is treated with a suitablerestriction enzyme to obtain fragments thereof, and is separated byelectrophoresis. The DNA is transferred onto a membrane likenitrocellulose, and is fixed to the membrane by ultraviolet lightirradiation or a like method.

Separately, a portion of the base sequence (preferably sequences ofabout 36-42 bases) of the F4 band DNA and that of the F10 band DNA aresynthesized, and they are labeled for detection. The base sequences arehybridized to the above DNA fixed onto the membrane, whereby the DNAcoming from a leishmania can be detected.

The labeling for the detection is conducted with a radioactive isotopesuch as ³² p (either at a 3'-terminal or at a 5'-terminal). Also Biotinlabeling with an avidin (or streptoavidin)-enzyme complex or haptenlabeling with an antibody-enzyme complex are useful for the detection.

Further, the detection can be conducted by dot hybridization (see Saiki,et al., Proc.Natl.Acad.Sci USA, 86, 6230-6234, 1989, or Japanese PatentLaid-Open No. 1-31496). The labeling substance is not specially limited,and fluorescent substances (fluorescein, fluorescein isothiocyanate,rhodamine, tetramethylrhodamine isothiocyanate, Texas Red,phycoerythrin, etc.), and chemiluminescent substances (acridine,Eu-DPTA, etc.) can be used preferably. An indirect method is applicable.Biotin, digoxigenin, and the like are also applicable.

In the present invention, for example, the detection can be conducted bybonding amino-link 2 (produced by ABI Co.) to 5'-terminal of thepolynucleotide to be detected.

Judgement of Infection and of Kind of Leishmania Infection

As described above, the F4-band DNA and F10-band DNA can be detected bygene amplification by use of a detecting polynucleotide primer of thepresent invention. Leishmania infection can be judged by observation ofF4, and brazil type leishmania infection can be judged by detection ofF10.

EXAMPLE

The present invention is described below in more detail by reference toexamples without limiting the invention by the examples.

(1) Extraction of Leishmania Protozoan Gene (Template Gene)

The investigation was conducted with the 7 kinds of protozoans below:

L. braziliensis panamensis (MHOM/PA/71/LS94)

L. braziliensis braziliensis (MHOM/BR/75/M2904)

L. braziliensis guyanensis (MHOM/BR/75/M4147)

L. mexicana mexicana (MNYC/BZ/62/M379)

L. mexicana aristedesi (MORY/PA/68/GML3)

L. mexicana amazonensis (MHOM/BR/73/M2269)

L. major (MHOM/SU/73/5ASKH)

L. chagasi (MHOM/BR/74/PP75)

The promastigotes of the leishmanias were respectively cultivated in aSchneider medium, recovered therefrom, and suspended in an SE buffersolution (0.15M NaCl, 0.1M EDTA (pH8.0)).

To the resulting suspension liquid, sarcosine was added to dissolve thecells, and Proteinase K was added thereto to allow the reaction toproceed at 60° C. for one hour.

After one hour, the reaction mixture was subjected to centrifugation at4° C. at 16,000 rpm for 90 minutes.

The supernatant was collected, and thereto an equal amount of alphenol-chloroform solution was added to remove protein.

Then a 2.5-fold amount of ethanol was added thereto, and the mixture wasleft standing at -80° C. for 20 minutes to al ow the DNA to precipitate.

Further, the mixture was subjected to centrifuge at 16,000 rpm for 10minutes. The precipitate was dissolved in a TE buffer solution (10 mMTris-HCl (pH8.0), 1 mM EDTP,) to obtain template DNAs.

(2) Primer for Gene Amplification Reaction

The six primfers below were synthesized as the arbitrary primers.

The synthesis was conducted by means of a DNA synthesizer (manufacturedby ABI Co.).

INV-1 (5'-ACCGGAATTCTTGGCTGTTGGCACGATGAG-3') SEQ ID NO:7

INV-2 (5'-TTGCAAGCTTTTATCTGGTTATATTGACAG-3') SEQ ID NO:8

LS-3 (5'-AAGTGTTGATACCCACTTTGT-3') SEQ ID NO:9

LS-2 (5'-CAATGGGTTACTGTTACAAC-3') SEQ ID NO:10

MS2-F (5'-CCTTAGGTTCTGGTAATGAC-3') SEQ ID NO:11

MS2-R (5'-GGGTGCAkTCTCACTGGGAC-3') SEQ ID NO:12

(3) Investigation on Conditions of Gene Amplification

To 50 ng of the template DNA prepared in (1), were added 250 ngrespectively of the above arbitrary primers, 15.6 μM of dNTP_(mix), and0.6 unit of Taq polymerase. Further thereto, 10 mM Tris-HCl (pH8.3), 50mM KCl, 1.5 mM MgCl₂, and 0.001% gelatin were added to the final volumeof 25 μL, and the gene amplification was conducted.

The conditions of the gene amplification reaction were as shown below.

(i) 94° C., 5 min: 1 cycle

(ii) 94°C., 1 min; 37° C., 1 min; 72° C., 1 min: 10 cycles

(iii) 94° C., 1 min; 60° C., 1 min; 72° C., 7 min: 30 cycles

(iv) 72° C., 7 min: 1 cycle

(v) Storage at 15° C.

After the gene amplification reaction, 5 μL of the reaction mixture wassubjected to 6%-polyacrylamide gel electrophoresis to separate theproducts, and the separated matters were stained by silver staining.

(4) Detection of Leishmania Protozoan Fragment

The results of (3) were studied with the primers of (2) as shown in FIG.1A and FIG. 1B.

F10 band characteristic to L.braziliensis, and F4 band characteristic toboth protozoans of L.brasiliensis and L.mexicana were detected by use ofthe primer of LS-3.

(5) Sequencing of Base of DNAS of F4-Band and F10-Band

The objective band portions of the polyacrylaminde gel were cut out, andthe bands were extracted (e.g., Molecular Cloning, Cold Spring HarborLaboratory Press, 2nd ed., 1989).

The DNAs of the respective extracted bands were inserted into a vectorby means of a TA cloning kit (Invitrogen Co.).

Sequencing of the DNA of the vector was conducted with M13 universalprimer and reverse primer by means of DNA Sequencer 373A (manufacturedby ABI Co.).

As the results, the base sequences shown in Sequence Table 1 andSequence Table 4.

F10 band DNA SEQ ID NO:1

CTGTGTTAAT CTCACGTCGTC CTTCTCTTCT CTTCTGACTG GCTCGGCGCT

CGGTACCGCT TCTCGTTTCG CTTTGAACGG GAGAGCGGAG GAGAACGAGG

AGGTGGGCGT ATCTG° CTGAT GAGAGCGGTC GGATCTGCAT GCATCACCGG

TCCCTCGGAT GCACACACAT ACACACACAC TCGGCCCGCA GTCCCTCGCT

TTGTGCCGCC TTTTTTCTTG TCTTGCCTTA CGCCATGTAC TGCGACCACC

CACACACACA CAC

F4 band DNA SEQ ID NO:4

GTTCATGCAC GCCACTACTT GCAAGGGTCA CTCGGCATTT TGCGAGGATA

AAGGGAAAGA GTTGACATTG CGGCGGAGGT TAGACATGCA AGTCAGGGCA

CGGATGTGCG CCATCTCGTA CCCTG

(6) Synthesis of Detecting Primers

From the base sequence obtained in (5), four base sequences, two from F4band and two from F10 band, were selected (SEQ ID NO:2 and 3, and SEQ IDNO:5 and 6), and were synthesized as the DNA-detecting primers.

The synthesis was conducted with a DNA synthesizer (manufactured by ABICo.).

(7) Amplification of DNA specific to Leishmania

The gene amplification reaction was conducted on the template DNAobtained in (1) with the F4 band-detecting primers (SEQ ID NO:5 and 6)and the F10 band-detecting primers (SEQ ID NO:2 and 3) synthesized in(6).

The reaction conditions were the same as in (3) except that the primerwas changed.

Specificalsy, to 50 ng of the template DNA prepared in (1), were added250 ng respectively of the above primers (combination of SEQ ID NO:5 and6, or combination of SEQ ID NO:2 and 3), 15.6 μM of dNTP_(mix), and 0.6unit of Taq polymerase. Further thereto, 10 mM Tris-HCl (pH8.3), 50 mMKCl, 1.5 mM MgCl₂, and 0.001% gelatin to adjust the final volume to 25μL, and the gene amplification was conducted.

The conditions of the gene amplification reaction were as shown below.

(i) 94° C., 5 min: 1 cycle

(ii) 94° C., 1 min. 60° C., 1 min; 72° C., 7 min: 30 cycles

(iii) 72° C., 7 min: 1 cycle

(iv) Storage at 15° C.

After the gene amplification reaction, 5 μL of the reaction mixture wassubjected to 6%-polyacrylamide gel electrophoresis to separate theproducts, and the separated matters were stained by silver staining.

With the F4-detecting primers (SEQ ID NO:5 and 6), amplification of F4band DNA was observed in all the kinds of leishmanias (FIG. 2A and FIG.2B).

With the F10-detecting primers (SEQ ID NO:2 and 3), amplification of F10band DNA was observed only in brazil type leishmanias (FIG. 3A and FIG.3B).

Furthermore, with a trypanosoma, a relative parasite, amplification ofDNA by F4 band-detecting primer and F10 baind-detecting primer wasinvestigated, and no amplification was caused (FIG. 4). Thus the aboveamplification was confirmed to be specific to the leishmania protozoans.

INDUSTRIAL APPLICABILITY

The present invention enables detection of DNA specific to leishmanias,isolation thereof, and identification of the base sequence thereof.

Since the leishmania protozoan can be detected precisely in a specimen,the present invention can be applied to a diagnosis reagent, a diagnosiskit, and the like for diagnosing the infection with the leishmaniaprotozoan. Further, since the brazil type and the mexico type ofleishmanias can be differentiated, useful judgement data can be obtainedfor treatment of leishmaniasis after leishmania infection has beenconfirmed. Furthermore, a reagent, a diagnosis kit, or the like fordiagnosis of many specimens quickly and simply at a time for large groupdiagnosis can be developed according to the present invention, forexample, for use in the area where leishmania is epidemic.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 12                                            - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 263 nucleic - # acids                                             (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - CTGTGTTAAT CTCAGTCGTC CTTCTCTTCT CTTCTGACTG GCTCGGCGCT CG - #GTACCGCT         60                                                                          - TCTCGTTTCG CTTTGAACGG GAGAGCGGAG GAGAACGAGG AGGTGGGCGT AT - #CTGCTGAT        120                                                                          - GAGAGCGGTC GGATCTGCAT GCATCACCGG TCCCTCGGAT GCACACACAT AC - #ACACACAC        180                                                                          - TCGGCCCGCA GTCCCTCGCT TTGTGCCGCC TTTTTTCTTG TCTTGCCTTA CG - #CCATGTAC        240                                                                          #               263CACA CAC                                                   - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 23 nucleic                                                        (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 #                23TCGG TAC                                                   - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 22 nucleic                                                        (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 #                 22TAA GG                                                    - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 125 nucleic - # acids                                             (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 - GTTCATGCAC GCCACTACTT GCAAGGGTCA CTCGGCATTT TGCGAGGATA AA - #GGGAAAGA         60                                                                          - GTTGACATTG CGGCGGAGGT TAGACATGCA AGTCAGGGCA CGGATGTGCG CC - #ATCTCGTA        120                                                                          #           125                                                               - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 26 nucleic                                                        (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 #              26  ACTT GCAAGG                                                - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 24 nucleic                                                        (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 #                24GCAC ATCC                                                  - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 30 nucleic                                                        (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 #           30     GTTG GCACGATGAG                                            - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 30 nucleic                                                        (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 #           30     GGTT ATATTGACAG                                            - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 21 nucleic                                                        (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 #21                TTTG T                                                     - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 20 nucleic                                                        (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                # 20               CAAC                                                       - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 20 nucleic                                                        (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                # 20               TGAC                                                       - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 20 nucleic                                                        (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                #  20              GGAC                                                       __________________________________________________________________________

We claim:
 1. An isolated polynucleotide comprising the sequence of SEQID NO:1.
 2. An isolated polynucleotide comprising the sequence of SEQ IDNO:2.
 3. An isolated polynucleotide comprising the sequence of SEQ IDNO:3.
 4. An isolated polynucleotide comprising the sequence of SEQ IDNO:4.
 5. A polynucleotide having a base sequence set forth in sequencelisting SEQ ID NO:5.
 6. A polynucleotide having a base sequence setforth in sequence listing SEQ ID NO:6.
 7. A method of detectingleishmania protozoan DNA in a sample comprising:specifically amplifyingsample DNA containing at least the nucleotide sequence of SEQ ID NO: 1or 4; and detecting the amplified DNA wherein the presence of amplifiedDNA containing SEQ ID NO: 1 or 4 is indicative of the presence ofleishmania protozoan DNA.
 8. A method of detecting leishmania protozoanDNA in a sample comprising:specifically amplifying sample DNA containingat least 15 contiguous nucleotides of SEQ ID NO: 1 or 4; and detectingthe amplified DNA wherein the presence of amplified DNA containing the15 contiguous nucleotides of SEQ ID NO: 1 or 4 is indicative of thepresence of leishmania protozoan DNA.